Mycoplasma sp. strain 56A97 was isolated from the liver of a 10-day-old Humboldt
penguin from a breeding colony at Chester Zoo. The Humboldt penguin is an
endangered species and breeding programmes have been established in zoos
worldwide. Initial testing of strain 56A97 indicated that it cross-reacted serologically
with Mycoplasma gallisepticum, an important respiratory pathogen of poultry.
Mycoplasma imitans, found in ducks and geese, also shows serological cross-reactions
with M. gallisepticum but it is a distinct species, although their 16S rRNA genes have
99.9% similarity.
Characterisation studies were undertaken to establish the taxonomic status of strain
56A97 to fulfil the minimum standards for description a new species within the class
Mollicutes. Electron microscopy confirmed that strain 56A97 lacked a rigid cell wall,
was bounded by a tri-laminar membrane and had an attachment organelle. It required
sterol and did not catabolise urea and could thus be assigned to the class Mollicutes,
order Mycoplasmatales, family Mycoplasmataceae genus Mycoplasma.
Strain 56A97 was serologically distinct from 107 recognised Mycoplasma spp. as
determined by growth inhibition and immunofluorescence. Metabolism inhibition
strongly suggested that it was distinct from M. gallisepticum and M. imitans. The 16S
rRNA gene of strain 56A97 was 99.6% similar to that of M. gallisepticum and 99.6%
similar to M. imitans. It was possible to distinguish between the three species without
DNA sequencing by restriction fragment length polymorphism (RFLP) of the 16S rRNA
genes. Furthermore a 400 base pair target within the rpoB of strain 56A97 was 91 -
92% similar to the same target in five M. gallisepticum strains, which themselves were
98-100% similar. The 16S-23S rRNA intergenic spacer region (ISR) of strain 56A97 was
72-73% similar to the five M. gallisepticum strains, whose similarity ranged from 96
100%. Phylogenetic analysis of the three genomic regions was performed. A strain
56A97-specific PCR was developed using a primer targeting the ISR.
Preliminary pathogenicity studies in chick embryo tracheal organ cultures (TOCs) and
embryonated chicken eggs showed that strain 56A97 caused ciliostasis in the TOCs and
mortality and stunting in embryos. Strain 56A97 DNA was detected in 82% of 101
healthy Humboldt penguins from eight collections and in 62% African penguins
(Spheniscus demersus) in another. Isolates were recovered from 14 healthy Humboldt
penguins and two African penguins. They were typed serologically and by RFLP as
strain 56A97. King and Rockhopper penguins, which belong to another genus, did not
appear to be infected despite co-habiting with infected penguins. In a parallel study
the 16S-23S rRNA ISR and a target within the mgc2 gene of M. gallisepticum were
amplified in 57 strains of M. gallisepticum and analysis of the results indicated a
potential value for strain differentiation and epidemiological investigations.